Project Summary Hyperpolarization-activated and cyclic nucleotide-gated ion channels (HCN) are highly expressed in the heart and central nervous system where they responsible for slowly activating currents that contribute to pacemaking activity. In addition to their role in generating rhythmic oscillations in neuronal circuits, these channels also play a crucial role in working memory and motor learning. They are important pharmacological targets for new drug development to treat disease conditions such as epilepsies and neuropathic pain. Despite the progress in understanding the structure and physiological role of these ion channels, there remains a significant gap in our knowledge of the biophysical mechanisms that underpin HCN channel behavior. These channels are unique in the voltage-gated ion channel superfamily and have the potential to provide new insights into inward rectification and ligand activation. For instance, ensemble ligand binding measurements using patch clamp fluorimetry have recently suggested a remarkable model of ligand activation that involves a sequence of positive and negative modulation of channel activity by physiological ligand. Although numerous crystal structures of cyclic nucleotide-binding domain (CNBD) from HCN channels are available, the mechanisms that underlie this unusual form of cooperativity remain unclear. The central goal of this project is to understand how the chemical structure and the resulting forces orchestrate ligand activation in HCN channels. This proposal takes advantage of the interdisciplinary expertise at UW-Madison to combine single molecule measurements of ligand binding with structural and functional analysis of ligand activation. We will test the hypothesis that ligand activation in HCN channels may involve a symmetry-breaking switch to a dimer of dimer configuration. In specific aim 1, we will use zero-mode waveguides to measure the binding of individual ligands to the cyclic-nucleotide binding domains. This will allow us to directly measure energetics of each ligand-binding step and to track the cooperativity associated with this process. With this analysis in hand, we will be able to identify the key molecular determinants responsible for each of the four ligands. In specific aim 2, we will use X-ray crystallography to determine the structures of the unliganded states of the HCN CNBDs as well as new conformations of their liganded forms. In specific aim 3, we will carry out functional analysis of ligand activation using electrophysiological and biochemical binding studies. These studies combined with mutagenesis will identify the molecular bases for isoform-specific differences in ligand activation. The proposed studies are expected to shed new light on the molecular forces that underlie conformational changes during the ligand activation in a voltage- and ligand-activated ion channel.